Diagnostic reagent composition for determining blood coagulation factors and method of use



United States Patent Ofifice 3,293,134 Patented Dec. 20, 1966 DIAGNOSTICREAGENT COMPOSITION FOR DE- TERMINING BLOOD COAGULATION FACTORS ANDMETHOD OF USE Jane G. Lenahan, Florham Park, and Monroe L. Miller andRaphael Cohen, Morristown, N.J., assignors to Warner-LambertPharmaceutical Company, Morris Plains, N.J., a corporation of Delaware NDrawing. Filed Sept. 8, 1961, Ser. No. 136,724

3 Claims. (Cl. 167-845) This invention relates to a diagnostic reagentcomposition for use in determining blood coagulant factors reduced bytreatment with anti-coagulants, particularly oral anti-coagulants usedin the treatments of humans, and to methods of blood plasma coagulationfactor determination therewith.

An object of the present invention is to provide a composite diagnosticreagent composition which is adapted for use without modification, otherthan aqueous reconstitution.

A further object of the invention is to provide such a diagnosticreagent composition and method which may be employed on patient plasmastored up to forty-eight hours or more.

Another object of the invention is to provide such a diagnostic reagentcomposition in admixed composite form as opposed ta the employment ofseparate individual components not in admixture prior to their admixturewith patient plasma.

Still another object of the invention is to provide a diagnostic reagentcomposition for controlling anti-coagulant therapy which will enabledetection in reduction of platelet factors in the blood plasma ofpatients under anti-coagulant therapy and simultaneously under treatmentwith other drugs, such as quinidine, which cause such reduction.

The present invention provides a novel composite diagnostic reagentcomposition and method for determining blood coagulant factors. Thepresent composition and method are sensitive to the extrinsiccoagulation system as well as to the usual intrinsic system, as measuredby standard techniques for controlling anticoagulant therapy, and may beemployed to test stored patient plasma. A tissue thromboplastin,preferably comprising aqueous extract -of acetone, treated horse brain,having weak activity toward human plasma is employed.

Adsorbed bovine plasma, treated to have undetectable concentrations ofthe factors which are known to be reduced in anti-coagulant treatment;namely, proconveitin (in the extrinsic system), Stuart factor (in boththe extrinsic and intrinsic system), prothrombin, is employed. Thisadsorbed bovine plasma is selected and treated to contain high,stabilizing concentrations of proaccelerin and fibrinogen. The mixtureof the thromboplastin and the plasma are buffered to a pH of 7.2 to 7.6to avoid variations in clotting activity due to departure from optimalpH. The mixture also includes calcium ion in sufficient amount foroptimal activity.

It has been discovered that the present diagnostic reagent composition,which is prepared to be free of added platelet factors, such as cephalinor substitute, provides sensitivity in the intrinsic system to thereduction of platelet factors by drugs, such as quinidine,simultaneously given with anti-coagulants, which characteristic is notknown to be heretofor available in any diagnostic reagent compositionfor controlling anti-coagulant therapy.

It is well recognized that a number of drugs used in the treatment ofheart disease may include as a side effect, thrombocytopenic purpura, ahemorrhagic condition caused by reduction in the number of bloodplatelets, which are the source for the platelet factor, an essentialconstituent of the intrinsic system of blood coagulation (Hunt J. C., etal., Proceedings of the Staff Meeting of the Mayo Clinic, vol. 33, No.4, p. 87 (February 1958). It is also recognized that treatment with suchdrugs, when the patient is on simultaneous anti-coagulation therapy, canresult in reduction of platelet factors which do not manifest themselvesas purpura, but which are clinically similar to the results ofoverdosage with anti-coagulants (Sussman, L. N., et al., JAMA, vol. 156,No. 7, p. 702, October 15, 1954).

There is commercially available a composite reagent comprisingthromboplastin from bovine, horse or swine brain having a low activitytoward human plasma; adsorbed bovine plasma; cephalin and calciumchloride, buffered to a pH of 6 to 8. Such composite diagnostic reagentcomposition is available in frozen-dried ampoule form. The employment ofthis composition is stated to permit a quantitative measure of thereduction of the plasma thromboplastin component (Christmas factor) byvirtue of the inclusion of the cephalin component. Thus, it has beenadvanced that this reagent composition enables simultaneous and combineddetermination of the coagulability of the blood with a single test whichis stated to proceed at the same rate for the specific factorsproconvertin, prothrombin, Stuart factor and Christmas factor.

However, it has not been established that reduction of the Christmasfactor is measurable at critically low levels of the order of 10% andbelow. On the other hand, the inclusion of cephalin is known tocontribute total insensitivity of the diagnostic reagent composition toreduction in platelet factors for which cephalin, by definition is asubstitute. The variable pH range of this material; namely, from pH 6 topH 8, which is employed with a weak thromboplastin for human plasma,renders the results subject to variability, due to departure from theoptimal pH of the blood clotting reaction which is established to be inthe immediate range of 7.2 to 7.6. It is believed that such a conditionmay cause results which simulate the action of the therapy when, infact, such results are due entirely to deficiencies in the reagentsystem. Thus, in effect, use of a diagnostic reagent composition of thetype having added cephalin can result in a masking of conditions causedby simultaneous therapy (in addition to anti-coagulant therapy). Due tothe masking of these conditions, simultaneous therapy may be continued,-

which in turn may give rise to hemorrhagic conditions, or the maskedcondition itself already may be hemorrhagic.

,As above mentioned, unitary compositions, including cephalin as acomponent, cannot be employed to detect reduction in platelet factors;on the other hand, the composition of the instant invention is adaptedfor use in detecting reduction of all those components effectivelymeasured by the commercially available composition.

Accordingly, the present invention provides a novel composite diagnosticreagent composition comprising tissue thromboplastin having weakactivity toward human plasma, adsorbed bovine plasma and calcium ion,buffered to a pH of 7.2 to 7.6, which is sensitive to changes in theextrinsic coagulation system, which also is sensitive to changes in theintrinsic coagulation system, as measured by standard methods, and whichalso is sensitive to components of the intrinsic system, such asplatelet factors, which may be reduced during anti-coagulant therapy bysimultaneous treatment with quinidine or other drugs. Other drugs whichexhibit thrombocytopenic purpura as side effects, when given to patientsunder anti-coagulant therapy, may cause reduction in the plateletfactors which are detectable by virtue of the present invention.

Thus, one of the primary features of the present composition is thedetection of reduction in platelet factors during anti-coagulant therapytreatment where the patient is additionally subjected to supplementaldrug treatment, as with quinidine, by means of a unitary one-stage testprocedure.

PREPARATION OF DIAGNOSTIC REAGENT COMPOSITION Example I 1 volume ofbarium sulfate adsorbed oxalated bovine plasma is added to 1 volume ofdistilled water, plus 1 volume of a solution which is 0.15 m. in respectto sodium chloride, and 0.25 m. with respect to calcium chloride, plus 1volume of aqueous extract of acetone treated horse brain tissue, whichhas been rendered 0.05 m. in respect to calcium chloride; plus, butterto bring the complete admixture to a pH of 7.2 to 7.6.

Example 11 METHOD OF USE Patient blood is obtained by veni-puncture andoxalated, the plasma being obtained by centrifuging. The sample then isimmediately transferred to a silicone-coated tube. Adequate portions ofthe reagent of Example I and the patient plasma are separately warmed to37 C. for three to five minutes, following which 0.05 ml. of patientplasma to be tested is added to 0.25 ml. of the reagent and the time forfibrin formation is noted. The fibrin formation time is compared with acurve plotted by diluting normal plasma to various levels withphysiological saline or water and the activity of the patient plasma asa percentage of normal activity is calculated by reference to theplotted curve.

' Percent activity Normal range 100 Therapeutic range -30 Patient bloodplasma obtained and stored as above and an adequate portion of thereagent of Example II are separately warmed at 37 C. for three to fiveminutes. 0.1 ml. of-the patient plasma to be tested is added to 0.5 ml.of the reagent and the time for fibrin formation is noted. The fibrinformation time is compared with a curve plotted by diluting normalplasma to various levels with physiological saline or water and theactivity of the patient plasma as a percentage of normal activity iscalculated by reference to the plotted curve.

Percent activity From the foregoing, it will be apparent that theinvention provides a novel composition and method for use in determiningblood coagulation factors and it will be apparent to those skilled inthe art that various changes may be made without departing from thespirit of the invention and therefore the invention is not limited towhat is described in the specification but only as indicated in theappended claims.

We claim:

1. A cephalin free diagnostic reagent composition for use in determiningblood coagulation factors in both extrinsic and intrinsic coagulationsystems having a pH of 7 .2-7.-6 and consisting essentially of (1)tissue thromboplastin having weak activity toward human plasma;

(2) adsorbed bovine plasma, free from Stuart factor, prothrombin,proconvertin and Christmas factor and containing proaccelerin andfibrinogen.

2. The diagnostic reagent composition of claim 1 wherein the weak tissuethromboplastin is an aqueous extract of acetone dried horse brain.

3. The method of determining blood plasma coagulation factor levelswhich comprises admixing patient plasma with the diagnostic reagentcomposition of claim 1 and measuring the length of time required to formfibrin.

Normal range Therapeutic range References Cited by the Examiner UNITEDSTATES PATENTS 4/ 1965 Owren 167--84.5

- OTHER REFERENCES Chemical Abst., vol. 46, 1952, p. 5168.

Dede: Am. J. Med. Tech., vol. 21, No. 4, 1955, pp. 222-231.

Hunt: Proceed. Staif Meeting of the Mayo Clinic, 3314, February 1958,pp. 87-92.

Langdell: The J. of Lab. and Clin. Med. 41:4, April 1953, pp. 637-647.

Mann: PSEBM, vol. 66, 1947, pp. 33-35.

Nature, vol. 186, April 9, 1960, pp. 173-174.

Owren: The Lancet, Nov. 7, 1959, pp. 754-758.

Rodman: Am. J. Clin. Path, vol 29, 1958, pp. 525-538. Stormorken: ActaPhysiol. Scand., vol. 41, 1957, pp. 301-305.

Sussmanr J.A.M.A., vol. 156, Oct. 15, 1954, pp. 702- 705.

Waaler: Scand. J. Clin. and Lab. Investigation, vol. 9, 1957, pp.322-330.

SAM ROSEN, Primary Examiner.

ANNA P. FAGELSON, Assistant Examiner.

1. A CEPHALIN FREE DIAGNOSTIC REAGENT COMPOSITION FOR USE IN DETERMININGBLOOD COAGULATION FACTORS IN BOTH EXTRINSIC AND INTRISIC COAGULATIONSYSTEMS HAVING A PH OF 7.2-7.6 AND CONSISTING ESSENTIALLY OF (1) TISSUETHROMBOPLASTIN HAVING WEAK ACTIVITY TOWARD HUMAN PLASMA; (2) ADSORBEDBOVINE PLASMA, FREE FROM STAUART FACTOR, PROTHROMBIN, PROCONVERTIN ANDCHRISTMAS FACTOR AND CONTAING PROCCELERIN AND FIGRINOGEN.